Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nucl ear localization signal-bearing proteins, and functions as an adapter to ac cess the importin beta-dependent impart pathway. In contrast to what is fou nd for importin beta, several isoforms of importin alpha, which can be grou ped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha 7. To analyze specific fun ctions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xen opus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin or proteins from humans or Xenopus bind their import receptor (im portin beta) and their export receptor (CAS) with only marginal differences . Using an in vitro import assay based on permeabilized HeLa cells, we comp ared the import substrate specificities of the various importin alpha prote ins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha 3, whereas most of the other substrates were imported by all importin alpha proteins with s imilar efficiencies. However, strikingly different substrate preferences of the various importin at proteins were revealed when two substrates were of fered simultaneously.